Pyridazino 4&#39;,5&#39;:3,4!pyrrolo- 2,1-a!-isoquinolines and the use thereof for preparing pharmaceutical preparations

ABSTRACT

The invention relates to new pyridazino[4&#39;,5&#39;:3,4]-pyrrolo[2,1-a]isoquinolines of the formula    &lt;IMAGE&gt;  (I)  and the physiologically acceptable salts thereof with acids and complex-forming agents, wherein X is O, S or NHO and R1, R3, R4, R5, R6, R7, R8 and R9 are defined as in the specification, and pharmaceutical preparations containing these compounds.

This is a continuation of application Ser. No. 08/360,863, filed Dec.21, 1994, now abandoned.

From German Patent Applications DE 35 00 941.1 and DE-35-25-048-8,cardiotonically active 9-amino-pyridozino 4',5':3,4! pyrrolo-2,1-a!isoquinolines are known. It is known from European PatentApplication No. 252-299(A) that these compounds have cardio- andneuroprotective effects and additionally promote blood circulation tothe tissues and the supply of oxygen to the tissues in the centralnervous system.

The invention relates to new pyridazino 4',5':3,4!-pyrrolo2,1-a!isoquinolines of formula I ##STR2## and the physiologicallyacceptable salts thereof with adds and complex-forming agents. The newcompounds have valuable therapeutically useful properties. They may beused as cardioprotective agents, as cerebroprotective agents(particularly for treating patients who have suffered a stroke or are indanger of suffering a stroke) and as agents for treating chronicallyinflammatory processes (e.g. bronchial asthma and arthritis). Thesecompounds may also be used as agents with an antiproliferative effectand as agents for treating ulcerative colitis and Crohn's disease.

In formula I:

X represents O,S or NHO;

R₁ has one of the following meanings:

a) a heterocyclic 5- or 6-membered ring containing a nitrogen atom andoptionally, as a further heteroatom, an oxygen, nitrogen or sulphuratom;

b) C₃₋₇ -cycloalkyl;

c) a straight-chained or branched, saturated or unsaturated alkyl grouphaving 1 to 10 or 2 to 10 carbon atoms, which may be substituted byhydroxy, C₁₋₄ -alkoxy, halogen, NH₂, NH-alkyl having 1 to 2 carbonatoms, N,N-di(C₁₋₂)alkylamino, NH-acyl having 2 to 4 carbon atoms, 1 or2 C₃₋₇ -cycloalkyl groups, phenoxy, 1 or 2 phenyl groups (wherein thephenyl ring or rings or phenoxy may in turn be mono- or disubstituted byhalogen, CF₃, C₁₋ -alkyl, C₁₋₂ -alkoxy, NH-alkyl having 1 to 2 carbonatoms, N,N-dialkyl having 1 to 2 carbon atoms, NH₂, N-acyl having 2 to 3carbon atoms, --OCH₂ O, alkylsulphonylamino, phenoxy or benzyloxy),furyl, thienyl, a nitrogen-containing heterocyclic 5- or 6-membered ringwhich may optionally contain an oxygen or sulphur atom as a furtherheteroatom (wherein the ring may optionally be substituted by C₁₋₄-alkyl);

R₃, R₄ and R₅, which may be identical or different, represent hydrogenor a C₁₋₄ -alkyl group;

R₇ and R₈, which may be identical or different, represent hydroxy; C₁₋₄-alkoxy; or C₁₋₄ -alkylthio;

R₆ and R₉, which may be identical or different, represent hydrogen;hydroxy; C₁₋₄ -alkoxy; C₁₋₄ -alkylthio; or 2 adjacent substituents ofthe substituents R₆, R₇, R₈ and R₉ together form the group --O--(CH₂)₁or 2 --O-- and the other 2 substituents are as hereinbefore defined.This excludes compounds of formula I as hereinbefore defined wherein XR₁represents the group SCH₃. These compounds are known as startingcompounds for preparing similar compounds (DE 35 00 941).

Particular mention should be made of compounds of formula I wherein X isas hereinbefore defined,

R₁ has one of the following meanings:

a) a heterocyclic 5- or 6-membered ring containing a nitrogen atom andoptionally, as a further heteroatom, an oxygen, nitrogen or sulphuratom;

b) C₃₋₇ -cycloalkyl;

c) a straight-chained or branched, saturated or unsaturated alkyl grouphaving 1 to 5 or 2 to 5 carbon atoms, which may be substituted byhydroxy, C₁₋₄ -alkoxy, halogen, NH₂, NH-alkyl having 1 to 2 carbonatoms, N,N-di(C₁₋₂)alkylamino, NH-acyl having 2 to 4 carbon atoms, C₃₋₇-cycloalkyl, phenyl (wherein the phenyl ring may in turn be mono- ordisubstituted by halogen, C₁₋₂ -alkyl, C₁₋₂ -alkoxy, NH-alkyl having 1to 2 carbon atoms, N,N-dialkyl having 1 to 2 carbon atoms, NH₂, N-acylhaving 2 to 3 carbon atoms or (C₁ or C₂) alkylsulphonylamino), furyl,thienyl, a nitrogen-containing heterocyclic 5- or 6-membered ring whichmay optionally contain as a further heteroatom an oxygen or sulphur atom(whilst the ring is optionally substituted by C₁₋₄ -alkyl);

R₃, R₄ and R₅, which may be identical or different, represent hydrogenor a C₁₋₄ -alkyl group;

R₇ and R₈, which may be identical or different, represent hydroxy; C₁₋₄-alkoxy; or C₁₋₄ -alkylthio and

R₆ and R₉, which may be identical or different, represent hydrogen;hydroxy; C₁₋₄ -alkoxy; C₁₋₄ -alkylthio.

Particular mention should also be made of compounds (I) wherein R₁ is astraight-chained or branched C₁₋₄ -alkyl group which is substituted byC₃₋₇ -cycloalkyl, thienyl or 1 or 2 unsubstituted phenyl groups or by asubstituted phenyl group the substituent(s) of which is or are definedas in claim 1 or 2, particularly those

wherein R₁ is (C₁₋₄)alkylcylcyclohexyl, preferably --CH₂ --C₆ H₁₁, or

wherein R₁ is (C₁₋₄)alkylphenyl, wherein the phenyl group isunsubstituted or is mono- or disubstituted by F, Cl, CF₃, methyl, ethyl,methoxy or ethoxy.

Particular mention should be made of compounds (I) wherein R₁ is one ofthe following groups: ##STR3## particularly those wherein R₁ is one ofthe following groups: ##STR4##

Mention should also be made of compounds (I) wherein R₃, R₄, R₅, R₆ andR₉ represent hydrogen and R₇ and R₈ represent C₁₋₄ -alkoxy or R₇ and R₈together represent --OCH₂ O-- or --OCH₂ CH₂ O--, particularly thosewherein

R₇ and R₈ represent methoxy.

Particular mention should be made of compounds (I) wherein X denotes O,S or NHO, R₃, R₄, R₅, R₆ and R₉ are hydrogen, R₇ and R₈ represent C₁₋₄-alkoxy or R₇ and R₈ together represent --OCH₂ O-- or --OCH₂ CH₂ O-- andR₁ represents ##STR5## --(CH₂)₁ or 2 CH(C₆ H₅)₂, wherein R₁₃ is CF₃,C(CH₃)₃ or --OCH₂ C₆ H₅ and y is 1 or 2, or the physiologicallyacceptable salts thereof with adds or complex-forming agents,particularly those wherein R₁ represents the group ##STR6##

wherein R₁₃ and y are as hereinbefore denned and/or

wherein R₇ and R₈ represent methoxy and/or

R₁ has one of the following meanings ##STR7## particularly those whereinR₁ has one of the following meanings: ##STR8## preferably wherein R₁ hasone of the following meanings: ##STR9##

Also preferred are those compounds of general formula I wherein R₁denotes a straight-chained or branched C₁₋₅ -alkyl group; methoxy-C₁₋₄-alkyl; cyclopropyl; cyclopentyl; cyclohexyl; cyclopropylmethyl;cyclohexylmethyl; phenylethyl, wherein the phenyl ring may optionally bemono- or disubstituted by methoxy, CF₃ or halogen; propargyl;(furan-2-yl)methyl; thienylmethyl; 2-hydroxyethyl; (pyridin-4-yl)-ethyl;benzyl; 3,3-diphenylpropyl; (thien-3-yl)ethyl; 4-phenylbutyl;

and

R₇ and R₈ independently of each other represent hydrogen; methyl;methoxy; hydroxy; or methylthio

and

R₃, R₄, R₅, R₆ and R₉ represent hydrogen.

The compounds of formula I are bases and may be converted in the usualway with inorganic or organic adds and salt and complex-forming agentsinto any desired physiologically acceptable adducts (salts).

Acids suitable for salt formation include, for example, hydrochloricacid, hydrobromic acid, hydriodic acid, hydrofluoric acid, sulphuricacid, phosphoric acid, nitric acid, acetic acid, propionic acid, butyricacid, caproic acid, valeric acid, oxalic acid, malonic acid, succinicacid, maleic acid, fumaric acid, lactic acid, tartaric acid, citricacid, malic acid, benzoic acid, p-hydroxybenzoic acid, phthalic acid,cinnamic acid, sallcylic acid, ascorbic acid, methanesulphonic acid andthe like.

Preferred compounds of general formula I are those wherein R₃, R₄, R₅,R₆ and R₉ represent hydrogen and R₇ and R₈ represent methoxy, and/or

R₁ represents a group --(CH₂)₀₋₅ --A, wherein A is

cyclopentyl, cyclohexyl, phenyl, phenyl which is mono- or disubstitutedby F, Cl, CH₃, CF₃, OCH₃ or OC₂ H₅, ##STR10##

The new compounds may be prepared by methods known per se.

The new compounds of formula I wherein X represents NHO may be obtainedby reacting a compound of general formula II ##STR11## wherein thegroups R₃, R₄, R₅, R₆, R₇, R₈ and R₉ are as hereinbefore defined, with acompound of general formula III

    H.sub.2 N--OR.sub.1                                        (III)

wherein R₁ is as hereinbefore defined.

A starting compound of general formula II is dissolved in a high boilinginert solvent, such as dimethylformamide, dimethylacetamide,chlorobenzene or hexamethylphosphoric acid triamide and refluxed withthe amine component of general formula III until the reaction has ended.The reaction time is between about 1 and 15 hours and depends on thestarting components used.

In the case of reactive hydroxylamines, alcohols or tetrahydrofuran mayalso be used as solvent; under certain circumstances it may beadvantageous to carry out the reaction in an autoclave.

If the hydroxylamines used are liquid and sufficiently high-boiling, thereaction may also be carried out in an excess of the amine without anyadditional solvent (e.g. in the case of o-benzylhydroxylamine),optionally under a nitrogen atmosphere.

In some cases it may be possible to use a reactant which also acts as asolvent during the reaction.

The products of general formula I wherein X represents S or O may beobtained by reacting a compound of general formula IV ##STR12## whereinthe groups R₃, R₄, R₅, R₆, R₇, R₈ and R₉ are as hereinbefore defined andZ represents oxygen or sulphur, with an alkylating reagent of formula V

    R.sub.1 --Y                                                V

wherein R₁ is as hereinbefore defined and Y denotes an anionic leavinggroup, e.g. Cl, Br, I, the methanesulphonic acid group, thetrifluoromethane-sulphonic acid, p-toluenesulphonic acid,p-nitrobenzenesulphonic acid or p-bromobenzenesulphonic acid group.However, the alkylating agent may also consist of other reagents whichare capable of transferring carbocations, e.g. "onium" compounds such asMeerwein salts, e.g. triethyloxonium-tetrafluoroborate, -phosphate or-hexachloroantimonate.

A starting compound of general formula IV is reacted with the alkylatingagent of general formula V in an inert solvent, e.g. dimethylacetamide,hexamethylphosphoric acid triamide, chlorobenzene or acetone. Thereaction is usually carried out at ambient temperature, occasionally atreflux temperature depending on the reactivity of the alkylating agent.The reaction time is between about 1 and 20 hours and depends on thestarting components used.

The pyridazino-pyrrolo-isoquinolines (I) according to the invention arebases and may be converted in the usual way into any desiredphysiologically acceptable add addition salts with inorganic or organicadds.

Acids suitable for salt formation include, for example, hydrochloricacid, hydrobromic acid, hydriodic acid, hydrofluoric acid, sulphuricacid, phosphoric acid, nitric acid, acetic acid, propionic acid, butyricacid, caprole acid, valetic acid, oxalic acid, malonic acid, succinicacid, maleic acid, fumaric acid, lactic acid, tartaric acid, citricacid, malic acid, benzoic acid, p-hydroxybenzoic acid, p-aminobenzoicacid, phthalic acid, cinnamic acid, salicylic acid, ascorbic acid andmethanesulphonic acid.

EXAMPLES

1. 5,6 -Dihydro-2,3-dimethoxy-9-(O-benzyl)-hydroxylaminopyridazino4',5':3,4!pyrrolo 2,1-a!-isoquinoline hydrochloride

3 g of 5-methyl compound, 5 g of o-benzylhydroxylamine hydrochloride and50 ml of toluene are refluxed for about 5 hours. After the reaction hasended (monitored by TLC) the mixture is cooled and the reaction productis suction filtered.

It is washed twice with toluene and divided between CH₂ Cl₂ and diluteNaOH. The organic phase is washed several times with water, dried overNa₂ SO₄ and evaporated down. The residue is taken up in a little CH₂Cl₂, optionally after purification over a silica gel column (eluant CH₂Cl₂ /MeOH=100+10 V.V.) and converted into the hydrochloride by theaddition of ethanolic HCl.

Yield 2.86 g (71.5% of theory).

2. 5,6-Dihydro-2,3-dimethoxy-9-(4-bromobenzyl)-mercaptopyridazino4',5':3,4!pyrrolo 2,1-a!-isoquinoline

1.28 g of 5,6-dlhydro-2,3-dimethoxypyridazino- 4',5':3,4!pyrrolo2,1-a!isoquinolin-9-(10H)thione are suspended in 30 ml ofdimethylacetamide and at ambient temperature 3.50 g of4-bromobenzylbromide are added with stirring. After about 20 minutes aclear, reddish-orange solution is formed from which orange crystals areprecipitated as stirring continues at ambient temperature. The mixtureis left to stand for 16 hours at ambient temperature, the crystals aresuction filtered and dissolved in a mixture of methylene chloride andmethanol (100+20). The mixture is washed first with dilute NaOH, thenwith water, dried over anhydrous sodium sulphate and evaporated down.The residue is crystallised from CH₂ Cl₂ :MeOH (100+20).

Yield 5.5 g (89.3% of theory).

3. 5,6-Dihydro-2,3,9-trimethoxypyridazino 4',5':3,4!-pyrrolo2,1-a!isoquinoline

A suspension of 6.3 g of 5,6-dihydxo-2,3-dimethoxypyridazino4',5':3,4!pyrrolo 2,1-a!-isoquinoline in 50 ml of dimethylacetamide isreacted at ambient temperature with 5 ml of freshly distilledmethyliodide. After about 1 hour a dear solution is formed. This isstirred for a further 30 hours at 50° C., left to cool and then theyellow crystals precipitated are suction filtered and dissolved in amethylene chloride/methanol mixture (100+20). This is washed first withdilute NaOH and then with H₂ O. The organic phase is dried overanhydrous Na₂ SO₄ and concentrated by evaporation. The residue isdissolved in a CH₂ Cl₂ --MeOH mixture (100+20) and crystallised by theaddition of ether.

Yield: 4.7 g (75.6% of theory) m.p.:>270° C.

The following Tables list compounds according to the invention which canbe prepared analogously to the above Examples.

                  TABLE 1                                                         ______________________________________                                         ##STR13##                                                                    Y = CH.sub.3                                                                  C.sub.2 H.sub.5                                                                ##STR14##                                                                     ##STR15##                                                                     ##STR16##                                                                    ______________________________________                                    

                                      TABLE 2                                     __________________________________________________________________________     ##STR17##                                                                    __________________________________________________________________________    Y =                                                                              CH.sub.3 (Mp. > 270° C.)                                                                ##STR18##                                                    C.sub.2 H.sub.5                                                                                ##STR19##                                                    CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.3                                                       ##STR20##                                                    CH.sub.2CHCH.sub.2                                                            CH.sub.2CCH                                                                                    ##STR21##                                                     ##STR22##                                                                                     ##STR23##                                                     ##STR24##                                                                                     ##STR25##                                                     ##STR26##                                                                                     ##STR27##                                                     ##STR28##                                                                                     ##STR29##                                                     ##STR30##                                                                                     ##STR31##                                                     ##STR32##                                                                                     ##STR33##                                                     ##STR34##                                                                                     ##STR35##                                                     ##STR36##                                                                                     ##STR37##                                                     ##STR38##                                                                                     ##STR39##                                                                     ##STR40##                                                                     ##STR41##                                                                     ##STR42##                                                                     ##STR43##                                                                     ##STR44##                                                 __________________________________________________________________________

                                      TABLE 3                                     __________________________________________________________________________     ##STR45##                                                                    __________________________________________________________________________    Y =                                                                              CH.sub.3                                                                                             ##STR46##                                              C.sub.2 H.sub.5                                                                                      ##STR47##                                              CH.sub.2CH(CH.sub.3).sub.2                                                    (CH.sub.2).sub.9CH.sub.3                                                                             ##STR48##                                              CH.sub.2CHCH.sub.2                                                                                   ##STR49##                                              CH.sub.2CCH                                                                   CH.sub.2CH.sub.2OC.sub.2 H.sub.5                                                                     ##STR50##                                               ##STR51##                                                                                           ##STR52##                                               ##STR53##                                                                     ##STR54##                                                                                           ##STR55##                                               ##STR56##                                                                     ##STR57##                                                                                           ##STR58##                                               ##STR59##                                                                     ##STR60##                                                                                           ##STR61##                                               ##STR62##                                                                                           ##STR63##                                               ##STR64##                                                                                           ##STR65##                                               ##STR66##                                                                                           ##STR67##                                                                     ##STR68##                                           Y =                                                                               ##STR69##                                                                     ##STR70##                                                                     ##STR71##                                                                     ##STR72##                                                                     ##STR73##                                                                     ##STR74##                                                                     ##STR75##                                                                     ##STR76##                                                                     ##STR77##                                                                 __________________________________________________________________________                                Mp. (°C.)                                  __________________________________________________________________________     ##STR78##                  183-185                                            ##STR79##                  228                                                ##STR80##                  153-155                                            ##STR81##                  210                                                ##STR82##                  200-202                                           __________________________________________________________________________

The present invention relates to new 9-substituted pyridazino4',5':3,4!pyrrolo 2,1-a!isoquinolines and pharmaceutical preparationscontaining these compounds.

The present invention also relates to the use of these new compounds.

The compounds are beneficial for treating degenerative and necrotisingdiseases of the brain. Preventive treatment of patients at risk of suchdiseases is also possible. The effect of the compounds is not based onan improvement in circulation of the blood through the tissues. Thecompounds are thus suitable for a novel treatment for epilepsy andAlzheimer's disease and particularly for the treatment of patients whohave suffered a stroke or are at risk of suffering a stroke.

The present invention further relates to the use of the above compoundsfor preparing agents for the treatment of chronic inflammatoryprocesses, ulcerative colitis and Crohn's disease and agents with anantiproliferative activity. The effect of the compounds can be explainedby their inhibition of the unselective cation channels (UCC).

The pathophysiology of chronic bronchial asthma is based on inflammatoryprocesses which are mediated by the activation of inflammatory cells.(BARNES, 1987; SEIFERT and SCHULTZ, 1991).

The receptor-regulated activation of inflammatory cells (e.g.neutrophilic granulocytes and mast cells or the permanent cell linesHL-60 cells or sensitised RBL cells, i.e. those charged withgammaglobulin E) is inhibited, irrespective of the nature of thestimulating agonists (e.g. endothelin, PAF, leukotrienes, chemotacticalpeptide fMLP or antigen against sensitised mast cells) by blockers ofunselective cation channels (UCC) (RINK, 1990). Through these channelsextracellular calcium, which is responsible for the persistence ofreceptor-mediated cell activations, enters the cells (PUTNEY, 1990). Ifthis supply of calcium is interrupted, a blockade of the activation ofinflammatory cells results.

Conventional calcium antagonists of the dihydropyridine orphenylalkylamine type do not inhibit either UCCs or inflammatoryprocesses (WELLS et al., 1986).

As a measurement of the cell activation or as a measurement of theinhibition thereof by UCC blockers, the kinetics of the cytoplasmiccalcium ion concentration in fura-2-charged cells is quantifiedfluorometrically using the method described by GRYNKIEWICZ et al.(1985). This procedure has proved a reliable screening method, withinthe scope of the invention, for detecting UCC blockers.

So-called functional THAPSIGARGIN inhibition has proved suitable for thespecific characterisation of blockers of the unselective cationchannels. THAPSIGARGIN is a rumour promoter described by THASTRUP et al.(Proc. Natl. Acad. Sci. (USA), 87, 2466-2470, 1990) which selectivelyand irreversibly inhibits the Ca²⁺ -ATPase of intracellular IP₃-sensitive Ca²⁺ -stores. Consequently the Ca²⁺ -stores are rapidlydepleted. As described by J. PUTNEY (Calcium, 11, 611-624, 1990) thedepletion of these stores constitutes the physiological stimulation foropening up unselective cation channels in the cell membrane. The resultof this is a massive influx of Na⁺ and Ca²⁺ into the cell. Because ofthese properties, Thapsigargin is suitable as an indirect stimulator foragonist- and IP₃ -independent opening up of the unselective cationchannels.

Within the scope of the present invention the Thapsigargin stimulationof unselective cation channels has been carried out successfully on HL60 cells (human leukaemia cells), on hippocampal and cortical neuronecells and on RBL-cells (rat basophilic lymphoma cells) and in this waythe existence of these channels in particular cell lines wasdemonstrated.

The cytoplasmic Ca²⁺ concentration ( Ca²⁺ !_(i)) plays an important partin the cell proliferation and in turnout growth (for a summary see L. R.ZACHARSKI, Journal of Medicine 19: 145-177, 1988). In particular, theCa²⁺ -influx into the cell stimulated by receptor activation withconsecutive inositoltriphosphate-(IP₃ -)-mediation would appear to be ofcrucial importance for oncogenic cell proliferation (U. KIKKAWA and Y.NISHIZUKA, Ann. REV. CELL. BIOL. 2: 149-178, 1986). This mechanism alsoplays a part in the formation of metastases and in "Multi-DrugResistance". (For a summary see the above-mentioned publication by L. R.ZACHARSKI, J. MED. 19: 145-177, 1980).

This hypothesis is supported by the fact that Thapsigargin, as anindirect stimulator of the unselective cation channels (UCC) not onlyleads to a Ca²⁺ -overload in the cell but is also a highly effectiveturnout promoter. (V. THASTRUP et al. Proceedings of the NATL. Acad.Sci: (USA) 87: 2466-2470, 1990). The blockade of the Ca²⁺ -influx by theUCC leads to normalisation of the intracellular Ca-ion concentration andhence to inhibition of rumour growth etc.

Conventional calcium antagonists do not inhibit these UCC. It has beenfound, surprisingly, that the compounds according to this inventioninhibit the influx of calcium into the cell through the UCC.

As shown by S. H. MURCH et al. (Lancet 339: 381-385, 15. February 1992)endothelin I plays an important pathophysiological role in inflammatoryintestinal diseases such as ulcerative colitis and Crohn's disease.Using immunohistochemical methods it has been shown that patients withCrohn's disease in the region of the submucosa and patients withulcerative colitis in the region of the lamina propria of the epitheliumof the large intestine show significantly and greatly increasedconcentrations of endothelin I compared with healthy normal people. Itis assumed that the local secretion of endothelin causes massivevasospasms with consecutive disseminated ischaemia with microinfarctswhich are regarded as the actual cause of the above diseases. Thevasospasmogenic effectiveness of endothelin is explained by a Ca²⁺-overload of vascular myocytes. Endothelin primarily triggers an IP₃-mediated intracellular release of Ca²⁺ which is followed by a massivetransmembranal Ca²⁺ -entry through dihydropyridine-insensitive channels.(M. S. Simonson et al. Clin. Invest. Med. 14: 499-507, 1991; T. Masakai,J. Cardiovasc. Pharmacol. 13:Suppl. 5, S1-S4, 1989; D. W. Hay, R. J.Phamacol. 100: 383-392, 1990). These channels are unselective cationchannels which have also been briefly described as existing in cells ofthe large intestine mucosa. (Chr. Siemer and H. Gogelein, Europ. J.Physiol. 420: 319-328, 1992).

The endothelin-stimulated activation of fura-2-charged human leukaemiacells (HL 60 cells) has proved a suitable screening model for detectingfunctional endothelin antagonists. In conformity with G. GRYNKIEWICZ etal. (J. Biol. Chem. 260:3440-3450, 1985) the intracellular Ca²⁺-concentration in the cytoplasm of HL 60 cells (suspensions) can bemonitored by spectrofluorometry and quantified as a measurement of cellactivation by endothelin. The stimulation was effected by adding 0.1 mMendothelin and could be inhibited in a dosage-dependent manner by meansof the substances according to the invention.

The functional endothelin antagonism of the substances according to theinvention is mediated through a blockade of the unselective cationchannels. Consequently, detection of a functionalThapsigargin-antagonism on RBL-hm1 cells is also a suitable screeningmethod for functional endothelin antagonists.

Carrying out the investigation:

For screening purposes, fura-2-charged adhesive RBL-hm 1 cells arestimulated with 0.1 mM Thapsigargin in a Ca²⁺ -free incubation medium.After 4 minutes, extracellular Ca²⁺ is restored to a concentration of1.5 mM and, using the fura-2-fluorescence, the excessive increase in thecytoplasmic Ca²⁺ -concentration caused by a massive transmembranal Ca²⁺-entry through unselective cation channels is recorded.

This entry is to be inhibited solely by unselective cation channelblockers in a dosage-dependent manner. Neither conventional calciumantagonists nor specific blockers of agonists which stimulate the IP₃-turnover are able to inhibit the transmembranal Ca²⁺ -entry triggeredindirectly by Thapsigargin. The compounds of the present invention aredistinguished by their inhibition of UCC.

The fluorometric calcium measurement in the cytoplasm of individualadhering RBL-hm1 cells is carried out analogously to the methoddescribed by KUDO and OGURA (1986) for neuronal cells. An AXIOVERT 35fluorescence microscope made by ZEISS is used in conjunction with animaging system made by HAMAMATSU, consisting of the ICMS-imageprocessing system, residual light camera with control unit and imageintensifier DVS 3000.

The kinetics of the cytoplasmic Ca²⁺ -concentration is recordedcontinuously as a concentration/time curve after the cell activationstimulated by Thapsigargin (0.1 μM). The curves of two activated cellcultures are compared in the presence and absence of 10 μM testsubstance. The area under these curves (area under the curve=AUC) isintegrated and recorded as a measurement of cell activation. Theinhibitory potency of the UCC-blockers tested is determined using thefollowing equation: ##EQU1##

% H=the percentage inhibition of the calcium entry through unselectivecation channels which is stimulated and inhibited by 10 μM of testsubstance.

AUC_(inh) =area under the curve recorded in the presence of thestimulant plus 10 μM inhibitory test substance.

AUC control=area under the curve which is recorded only after theaddition of the stimulant.

Literature relating to the above explanations:

BARNES P. J., I. W. RODGER and N. C. THOMSON Pathogenesis of asthma, in"ASTHMA, basic mechanisms and clinical management"

ED by P. J. BARNES; ACADEMIC PRESS, LONDON, 1988

GRYNKIEWICZ G., M. POENIE and R. Y. TSIEN

A new generation of Ca²⁺ -indicators with greatly improved fluorescenceproperties

J. BIOL. CHEM. 260: 3440-3450, 1985

HIDE, M. and M. A. BEAVEN

Calcium influx in a rat mast cell (RBL-2H3) line

J. BIOL. CHEM. 266 15221-15229, 1991

KUDO, Y. and A. OGURA

Glutmate-induced increase in intracellular Ca²⁺ -concentration inisolated hippocampal neurones

BR. J. PHARMACOL. 89: 191-198; 1986

PUTNEY, J. W., jr.

Capacitative Calcium entry revised

CELL CALCIUM 11: 611-624, 1990

RINK, T. J.

Receptor-mediated calcium entry

FEBS LETT. 268: 381-985, 1990

SEIFERT, R. and G. SCHULTZ

The superoxide forming NADPH oxidase of phagocytes: An enzyme systemregulated by multiple mechanism

REV. PHYSIOL. BIOCHEM. PHARMACOL., Vol. 117,

SPRINGER VERL., 1991

WELLS, E., C. G. JACKSON, S. T. HARPER, J. MANN and R. P. EAOY

Characterization of primate bronchoalveolar mast cells II, inhibition ofhistamine, LTC₄ and PGF_(2a) release from primate bronchoalveolar mastcells and a comparison with rat peritoneal mast cells

J. IMMUNOL. 137: 3941-3945, 1986.

Results of measurement:

The percentage inhibition of UCC after Thapsigargin stimulation (0.1 μMThapsigargin) in RBL-hm 1 cells is given. The concentration of the testsubstances is 10⁻⁵ mol or 10⁻⁸ mol).

                                      TABLE                                       __________________________________________________________________________    RPL-hm 1 cells - Thapsigargin (0.1 μM) stimulation                          ##STR83##                                                                                         % H (10.sup.-5 M)                                                                     % H (10.sup.-6 M)                                __________________________________________________________________________    R.sub.1 (X:S)                                                                  ##STR84##                   21.8                                              ##STR85##                   26.1                                              ##STR86##           61.6                                                      ##STR87##           62.9                                                      ##STR88##           81.9    34.7                                              ##STR89##           94.6    52.9                                              ##STR90##           97.5    56.1                                             CH.sub.3             46.4                                                     R.sub.1 (X:O)                                                                 CH.sub.3             62.5                                                     __________________________________________________________________________

The functional antiinflammatory effectiveness can be demonstrated bymeans of the following test:

Individual RBL-2H3-cells (a turnout cell line related to the mast cells)adhering to glass slides are used.

The cultivation and attachment of the RBL-2H3-cells are carried out bythe method described by HIDE and BEAVEN (1991). In order to sensitisethe adhesive RBL-2H3-cells the cells are incubated for 2 hours atambient temperature with a 1:2000 diluted commercial gammaglobulinE-solution against a dinitrophenol-bovine serum albumin complex(DNP-BSA-antigen). The cells are then washed. By the addition of 0.1 mlof DNP-BSA-solution (10 μg/ml) there is a massive immunological cellactivation which is mediated by a cytoplasmic Ca²⁺ -overload. Thefluorometric calcium measurement in the cytoplasm of individual adheringRBL-2H3-cells is carried out analogously to the method described by KUDOand OGURA (1986) for neuronal cells, which is also explainedhereinbefore in this specification.

The comparison used in these investigations is (10 μM) chromoglycatewhich brings about an approximately 50% inhibition of theantigen-induced cell activation.

In this test the above-mentioned compounds demonstrate % H values whichare comparable with the values specified hereinbefore.

Tests on microcultures of various human tumour cell lines using thetetrazolium assay in order to determine the antiproliferative effect ofthe substances according to the invention surprisingly showed that thecompound tested was 5 to 100 times more potent than the comparisonsubstance Verapamil.

The antiproliferative effectiveness of the test substances wasdetermined by means of the MTT test described by MOSMANN (J. IMMUNOL.METH. 65: 55-63, 1983), DENIZOT et al. (J. IMMUNOL. METH. 89: 271-277,1986) and J. ELIASON et al. (INT. J. CANCER 46: 113-117, 1990). (MTT=3--(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide! producedby CHEMICON Inc. El Segundo, Calif., USA). This indicator is metabolisedonly by living cells with intact mitochondria into a blue formazaneproduct. The following human tumour cell lines were used in our test: A549 (adenocarcinoma of the lung), A 431 (epidermal carcinoma of thevulva), PC 3 (adenocarcinoma of the prostate), SK BR 3 (adenocarcinomaof the breast), HT 29 (CX1 1) (adenocarcinoma of the colon) and K 562(chronic myeloid leukaemia cell).

The test was carried out on microtitre plates. Each well contained 100μl of a cell suspension (0.2×10⁶ cells per ml). The incubation mediumused was RPMI 1640 with 10% heat-inactivated foetal calves' serum and 50μg/ml of gentamycin. The cell suspensions were incubated for 0, 24, 48or 72 hours in air with a humidity at saturation point in a CO₂ (5%)/air(95%) mixture at 37° C., incubated in the presence and absence ofvariable concentrations of antiproliferative test substances. The testsubstances were dissolved in DMSO (final dilution: 0.1%). Then 10 μl ofMTT-solution (3 mg/ml) were added, followed after 3 hours by 100 μl ofan isopropanol solution containing 0.08N HCl. After a further hour, thelight absorption at 570 nm (comparative wavelength 630 nm) wasdetermined in a microplate reader. The light absorption is directlyproportional to the number of living cells. The half-maximum inhibitoryconcentrations of the substances tested were 1 μg/ml.

The vasospasmolytic effectiveness of the above-mentioned functionalendothelin and Thapsigargin antagonists were confirmed on an isolatedblood vessel preparation: coronary perfusion was continuouslyquantified, on retrogressively perfused, spontaneously beatingLANDENDORFF hearts taken from rats, by means of electromagnetic flowmeasurement (apparatus supplied by Hugo Sachs Elektronik, MARCH). Thismeasuring apparatus could be used to record the extent, duration andpattern of vascular spasms with a high degree of accuracy. If perfusionis carried out with 100 nM endothelin concentration, the coronaryperfusion flow is reduced from 11 to 5 ml/min. The restriction inperfusion can be reversed by means of the substances according to theinvention. The potencies of the compounds according to the inventionwith regard to Thapsigargin inhibition on fura-2-charged RBL-hm 1-cellsor the effectiveness of endothelin-inhibition on fura-2-charged HL 60cells correlates dearly with the vasospasmolytic effectiveness of thetest substances detected on the Langendorff preparation. It can beconcluded from this that, underlying the vasospasmolytic endothelinantagonism of the substances tested, there is a blockade of theunselective cation channels.

The compounds may be administered both enterally and parenterally. Thesuggested dose for oral use ranges from 0.1 to 500 mg of activesubstance per dose and, for intravenous use, from 0.05 to 150 mg perdose. The desired therapeutic dose depends on the indication and form ofadministration and can be determined experimentally.

Suitable forms include, for example, tablets, capsules, suppositories,solutions, syrups, emulsions, aerosols or dispersible powders. Tabletsmay be produced, for example, by ruling the active substance orsubstances with known excipients, e.g. inert diluents such as calciumcarbonate, calcium phosphate or lactose, disintegrants such as cornstarch or alginic add, binders such as starch or gelatine, lubricantssuch as magnesium stearate or talc and/or agents for obtaining delayedrelease, such as carboxypolymethylene, carboxymethylcellulose, celluloseacetate phthalate or polyvinylacetate. The tablets may also consist ofseveral layers.

Coated tablets may be produced analogously by coating cores made in thesame way as the tablets with substances conventionally used for tabletcoatings, e.g. collidone or shellack, gum arabic, talc, titanium dioxideor sugar. In order to obtain delayed release or avoid incompatibilities,the core may also consist of several layers. Similarly, the tabletcoating may consist of several layers to achieve delayed release, whilstthe excipients mentioned for the tablets may be used.

Syrups containing the active substances or combinations of activesubstances according to the invention may additionally contain asweetener such as saccharin, cyclamate, glycerol or sugar as well as aflavour enhancer, e.g. a flavouring such as vanillin or orange extract.They may also contain suspension adjuvants or thickeners such as sodiumcarboxymethylcellulose, wetting agents, e.g. condensation products offatty alcohols with ethylene oxide or preservatives such asp-hydroxybenzoates.

Injectable solutions are produced in the usual way, e.g. by addingpreservatives such as p-hydroxybenzoates or stabilisers such as alkalimetal salts of ethylene dismine tetraacetic add, and are thentransferred into injection vials or ampoules.

Capsules containing one or more active substances or combinations ofactive substances may be prepared for example by mixing the activesubstances with inert carriers such as lactose or sorbitol andencapsulating them in gelatine capsules.

Suitable suppositories may be produced for example by mixing withcarriers provided for this purpose, such as neutral fats orpolyethyleneglycol or derivatives thereof.

Processes for preparing the compounds of formula I are described inEuropean Patent Applications 252 299 and U.S. Pat. No. 5,614,516, towhich reference is hereby made.

Examples of Pharmaceutical Preparations

a) Coated tablets

1 tablet core contains:

    ______________________________________                                        Active substance of general formula I                                                                  30.0   mg                                            Lactose                  100.0  mg                                            Corn starch              75.0   mg                                            Gelatine                 3.0    mg                                            Magnesium stearate       2.0    mg                                                                     210.0  mg                                            ______________________________________                                    

Preparation

The active substance mixed with lactose and corn starch is granulatedwith a 10% aqueous gelatine solution through a 1 mm mesh screen, driedat 40° C. and rubbed through a screen once more. The granules thusobtained are mixed with magnesium stearate and compressed. The coresproduced in this way are coated in the usual manner with a coatingconsisting of an aqueous suspension of sugar, titanium dioxide, talc andgum arabic. The finished coated tablets are polished with beeswax.

b) Tablets

    ______________________________________                                        Active substance of general formula I                                                                  30.0   mg                                            Lactose                  100.0  mg                                            Corn starch              70.0   mg                                            Soluble starch           7.0    mg                                            Magnesium stearate       3.0    mg                                                                     210.0  mg                                            ______________________________________                                    

Preparation

The active substance and magnesium stearate are granulated with anaqueous solution of the soluble starch, the granules are dried andinternately mixed with lactose and corn starch. The mixture is thencompressed into tablets weighing 210 mg.

c) Capsules

    ______________________________________                                        Active substance according to formula I                                                                20.0   mg                                            Lactose                  230.0  mg                                            Corn starch              40.0   mg                                            Talc                     10.0   mg                                                                     300.0  mg                                            ______________________________________                                    

Preparation

The active substance, lactose and corn starch are first combined in amixer and then in a grinding machine. The mixture is returned to themixer, thoroughly combined with the talc and mechanically packed intohard gelatine capsules.

d) Tablets

    ______________________________________                                        Active substance according to the invention                                                             40.0   mg                                           Lactose                   100.0  mg                                           Corn starch               50.0   mg                                           Colloidal silica          2.0    mg                                           Magnesium stearate        3.0    mg                                           total                     195.0  mg                                           ______________________________________                                    

Preparation

The active substance is mixed with some of the excipients and granulatedwith a solution of the soluble starch in water. After the granules havedried the remaining excipients are added and the mixture is compressedto form tablets.

e) Coated tablets

    ______________________________________                                        Active substance according to the invention                                                             20.0   mg                                           Lactose                   100.0  mg                                           Corn starch               65.0   mg                                           Colloidal silica          2.0    mg                                           Soluble starch            5.0    mg                                           Magnesium stearate        3.0    mg                                           total                     195.0  mg                                           ______________________________________                                    

Preparation

The active substance and excipients are compressed to form tablet coresas described in Example a) 1 and these are then coated in the usual waywith sugar, talc and gum arabic.

f) Suppositories

    ______________________________________                                        Active substance according to the invention                                                             50.0   mg                                           Lactose                   250.0  mg                                           Suppository mass q.s. ad  1.7    g                                            ______________________________________                                    

Preparation

The active substance and lactose are mixed together and the mixture isuniformly suspended in the molten suppository mass. The suspensions arepoured into chilled moulds to form suppositories weighing 1.7 g.

g) Ampoules

    ______________________________________                                        Active substance according to the invention                                                             20.0   mg                                           Sodium chloride           5.0    mg                                           Twice distilled water q.s. ad                                                                           2.0    ml                                           ______________________________________                                    

Preparation

The active substance and the sodium chloride are dissolved in twicedistilled water and the solution is transferred under sterile conditionsinto ampoules.

h) Ampoules

    ______________________________________                                        Active substance according to the invention                                                             10.0   mg                                           Sodium chloride           7.0    mg                                           Twice distilled water q.s. ad                                                                           1.0    ml                                           ______________________________________                                    

i) Drops

    ______________________________________                                        Active substance according to the invention                                                            0.70    g                                            Methyl p-hydroxybenzoate 0.07    g                                            Propyl p-hydroxybenzoate 0.03    g                                            Demineralised water q.s. ad                                                                            100.00  ml                                           ______________________________________                                    

Preparation

The active substance and preservatives are dissolved in demineralisedwater, the solution is filtered and transferred into 100 ml vials.

What is claimed is:
 1. A compound of formula (I) ##STR91## wherein X isO, S or NHO;R₁ has one of the following meanings:a) C₃₋₇ -cycloalkyl; b)a straight-chained or branched alkyl group having 1 to 10 or an alkenylor alkynyl group having 2 to 10 carbon atoms, which alkyl, alkenyl oralkynl group may be substituted by hydroxy, C₁₋₄ -alkoxy, halogen, NH₂,NH-alkyl having 1 to 2 carbon atoms, N,N-di(C₁₋₂)alkylamino, NH-acylhaving 2 to 4 carbon atoms, 1 or 2 C₃₋₇ -cycloalkyl groups, phenoxy, 1or 2 phenyl groups (wherein the phenyl ring or rings or phenoxy may inturn be mono- or disubstituted by halogen, CF₃, C₁₋₄ -alkyl, C₁₋₂-alkoxy, NH-alkyl having 1 to 2 carbon atoms, N,N-dialkyl having 1 to 2carbon atoms, NH₂, N-acyl having 2 to 3 carbon atoms, --OCH₂ O--, (C₁ orC₂) alkylsulphonylamino, phenoxy or benzyloxy), furyl or thienyl; R₃, R₄and R₅, which may be identical or different, are hydrogen or a C₁₋₄-alkyl group; R₇ and R₈, which may be identical or different, arehydroxy; C₁₋₄ -alkoxy; or C₁₋₄ -alkylthio; R₆ and R₉, which may beidentical or different, are hydrogen; hydroxy; C₁₋₄ -alkoxy; C₁₋₄-alkylthio; or 2 adjacent substituents of the substituents R₆, R₇, R₈and R₉ together form the group --O--(CH₂)₁ or 2 --O-- and the other 2substituents are as hereinbefore defined, or a pharmaceuticallyacceptable salt thereof, with the exception of compounds of formula I ashereinbefore defined wherein XR₁ is the group SR₁ wherein R₁ is C₁₋₅alkyl or benzyl.
 2. A compound as recited in claim 1, wherein X is O, Sor NHO;R₁ has one of the following meanings:a) C₃₋₇ -cycloalkyl; b) astraight-chained or branched alkyl group having 1 to 5 or an alkenyl oralkynyl group having 2 to 5 carbon atoms, which which alkyl, alkenyl oralkynl group may be substituted by hydroxy, C₁₋₄ -alkoxy, halogen, NH₂,NH-alkyl having 1 to 2 carbon atoms, N,N-di(C₁₋₂)alkylamino, NH-acylhaving 2 to 4 carbon atoms, C₃₋₇ -cycloalkyl, 1 or 2 phenyl groups(wherein the phenyl ring or rings may in turn be mono- or disubstitutedby halogen, CF₃ C₁₋₄ -alkyl, C₁₋₂ -alkoxy, NH-alkyl having 1 to 2 carbonatoms, N,N-dialkyl having 1 to 2 carbon atoms, NH₂, N-acyl having 2 to 3carbon atoms, (C₁ or C₂) alkylsulphonylamino or benzyloxy), furyl orthienyl; R₃, R₄ and R₅, which may be identical or different, arehydrogen or a C₁₋₄ -alkyl group; R₇ and R₈, which may be identical ordifferent, are hydroxy; C₁₋₄ -alkoxy; or C₁₋₄ -alkylthio and R₆ and R₉,which may be identical or different, are hydrogen; hydroxy; C₁₋₄-alkoxy; C₁₋₄ -alkylthio, or 2 adjacent substituents of the substituentsR₆, R₇, R₈ and R₉ together form the group --O--(CH₂)₁ or 2 --O--and theother 2 substituents are as hereinbefore defined.
 3. A compound asrecited in claim 1, whereinR₁ has one of the following meanings:a) C₃₋₇-cycloalkyl; b) a straight-chained or branched alkyl group having 1 to 5or an alkenyl or alkynyl group having 2 to 5 carbon atoms, which whichalkyl, alkenyl or alkynl group may be substituted by hydroxy, C₁₋₄-alkoxy, halogen, NH₂, NH-alkyl having 1 to 2 carbon atoms,N,N-di(C₁₋₂)alkylamino, NH-acyl having 2 to 4 carbon atoms, C₃₋₇-cycloalkyl, phenyl (wherein the phenyl ring may in turn be mono- ordisubstituted by halogen, C₁₋₂ -alkyl, C₁₋₂ -alkoxy, NH-alkyl having 1to 2 carbon atoms, N,N-dialkyl having 1 to 2 carbon atoms, NH₂, N-acylhaving 2 to 3 carbon atoms or (C₁ or C₂) alkylsulphonylamino, furyl orthienyl; R₃, R₄ and R₅, which may be identical or different, arehydrogen or a C₁₋₄ -alkyl group; R₇ and R₈, which may be identical ordifferent, are hydroxy; C₁₋₄ -alkoxy; or C₁₋₄ -alkylthio and R₆ and R₉,which may be identical or different, are hydrogen; hydroxy; C₁₋₄-alkoxy; C₁₋₄ -alkylthio.
 4. A compound as recited in claim 1, whereinR₁ is a straight-chained or branched C₁₋₄ -alkyl group which issubstituted by C₃₋₇ -cycloalkyl, thienyl or 1 or 2 unsubstituted phenylgroups or by a substituted phenyl group.
 5. A compound as recited inclaim 4, wherein R₁ is (C₁₋₄) alkylcyclohexyl.
 6. A compound as recitedin claim 5, wherein R₁ is --CH₂ --C₆ H₁₁.
 7. A compound as recited inclaim 4, wherein R₁ is (C₁₋₄)alkylphenyl, wherein the phenyl group isunsubstituted or is mono- or disubstituted by F, Cl, CF₃, methyl, ethyl,methoxy or ethoxy.
 8. A compound as recited in claim 1, wherein R₃, R₄,R₅, R₆ and R₉ are hydrogen and R₇ and R₈ are C₁₋₄ -alkoxy or R₇ and R₈together are --OCH₂ O-- or --OCH₂ CH₂ O--.
 9. A compound as recited inclaim 8, wherein R₇ and R₈ are methoxy.
 10. A compound as recited inclaim 1, wherein X is O, S or NHO; R₃, R₄, R₅, R₆ and R₉ are hydrogen;R₇ and R₈ are C₁₋₄ -alkoxy or R₇ and R₈ together are --OCH₂ O-- or--OCH₂ CH₂ O-- and R₁ is ##STR92## --(CH₂)₁ or 2 CH(C₆ H₅)₂, wherein R₁₃is CF₃, C(CH₃)₃ or --OCH₂ C₆ H₅ and y is 1 or 2, or.
 11. A compound asrecited in claim 10, wherein R₁ is the group ##STR93## wherein R₁₃ and yare defined as in claim
 11. 12. A compound as recited in claim 10,wherein R₇ and R₈ are methoxy.
 13. A compound as recited in claim 1,wherein X is S.
 14. A compound as recited in claim 1, wherein R₁ is oneselected from the group consisting of: ##STR94##
 15. A compound asrecited in claim 1 wherein R₁ is selected from the group consisting of:##STR95##
 16. A compound as recited in claim 10, wherein R₇ and R₈ aremethoxy and R₁ is selected from the group consisting of: ##STR96##
 17. Acompound as recited in claim 1, wherein X is O, S or NHO; R₃, R₄, R₅, R₆and R₉ are hydrogen; R₇ and R₈ represent methoxy; and, R₁ is selectedfrom the group consisting of: ##STR97##
 18. A compound as recited inclaim 17, wherein R₁ is selected from the group consisting of: ##STR98##19. A compound as recited in claim 1, wherein X is O, S or NHO; R₃, R₄,R₅, R₆ and R₉ are hydrogen; R₇ and R₈ are methoxy; and R₁ is selectedfrom the group consisting of: ##STR99##
 20. A pharmaceutical compositioncomprising a compound in accordance with claim 1 and a pharmaceuticallyacceptable carrier.